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Received April 23, 2004
Revised May 18, 2004
Accepted after revision June 28, 2004
1 University of Washington
* To whom correspondence should be addressed. E-mail: hille{at}u.washington.edu.
We studied modulation of current in tsA-201 cells coexpressing rat erg1 channels with M1 muscarinic receptors. Maximal current was inhibited 30% during muscarinic receptor stimulation, with a small positive shift of the midpoint of activation. Inhibition was attenuated by coexpression of RGS2 or of a dominant-negative Gq, but not by N-ethylmaleimide or C3 toxin. Overexpression of a constitutively-active form of Gq (but not of G13 or of Gs) abolished the erg current. Hence, Gq/11 and not Gi/o or G13, likely mediates muscarinic inhibition. Muscarinic suppression of erg was attenuated by chelating intracellular Ca2+ to < 1 nM free Ca2+ with 20 mM BAPTA in the pipette, but suppression was normal if internal Ca2+ was strongly clamped to a 129 nM free Ca2+ level with a BAPTA buffer and this was combined with numerous other measures to prevent intracellular Ca2+ transients (pentosan polysulfate, preincubation with thapsigargin, and removal of extracellular Ca2+). Hence a minimum amount of Ca2+ was necessary for the inhibition but a Ca2+ elevation was not. The ATP analogue AMP-PCP did not prevent inhibition. The PKC blockers staurosporine and bisindolylmaleimide I did not prevent inhibition, and the PKC-activating phorbol ester PMA did not mimic it. Neither the tyrosine kinase inhibitor genistein nor the tyrosine phosphatase inhibitor dephostatin prevented inhibition by oxo-M. Hence protein kinases are not needed. Experiments with a high concentration of wortmannin were consistent with recovery being partially dependent on PIP2 resynthesis. Wortmannin did not prevent muscarinic inhibition. Our studies of muscarinic inhibition of erg current suggest a role of phospholipase C, but not the classical downstream messengers, such as protein kinase C or a calcium transient.
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